Digital imaging system for assays in well plates, gels and blots

ABSTRACT

An electronic imaging system is disclosed, for assessing the intensity of calorimetric, fluorescent or luminescent signal in a matrix consisting of wells, microwells, hybridization dot blots on membranes, gels, or other specimens. The system includes a very sensitive area CCD detector, a fast, telecentric lens with epi-illumination, a reflective/transmissive illumination system, an illumination wavelength selection device, and a light-tight chamber. A computer and image analysis software are used to control the hardware, correct and calibrate the images, and detect and quantify targets within the images.

This patent application claims the priority of U.S. ProvisionalApplication No. 60/024,043 filed Aug. 16, 1996.

This is a divisional, of application Ser. No. 9/240,649, filed Jan. 29,1999. Each of these prior applications is hereby incorporated herein byreference, in its entirety. Ser. No. 9/240,649, which was published inEnglish, was a continuation of International Application No.PCT/US97/15269 filed Aug. 12, 1997.

FIELD OF THE INVENTION

The present invention relates generally to assay analyzing systems and,more particularly, concerns a system and method for creating digitalimages of randomly arranged specimens (e.g. beads within gels, colonieswithin petri dishes) or specimens arranged in regular arrays (e.g. wellsin plastic plates, dots spotted onto membranes). The invention iscapable of creating digital images and performing automated analyses ofspecimens which emit very low levels of fluorescence, chemiluminescence,or bioluminescence. More particularly, the invention is designed for theanalysis of luminance arising from assays within well plates and gelmedia, and on membranes, glass, microfabricated devices, or othersupports.

BACKGROUND OF THE INVENTION Types of Assays

Many chemical and molecular biological assays are designed so thatchanges in the absorbance, transmission, or emission of light reflectreactions within the specimen. Therefore, instruments used to quantifythese assays must detect alterations in luminance.

Wells. Some assays are conducted within discrete flasks or vials, whileothers are performed within plastic plates fabricated to contain anumber of regularly spaced wells. “Well plate” assays are higher inthroughput and lower in cost than similar assays in discrete containers.Standard well plates contain 96 wells in an area of 8×12 cm. The trendis to higher numbers of wells, within the same plate size. Today'shighest commercial density is 384 wells. Very high density arrays ofsmall wells (microwells, e.g. thousands/plate with a fill volume of lessthan 1 ul/well) are under development, and will become commerciallyavailable as microwell filling and detection technologies mature.

Dot blots. Grids of small dots (reactive sites) are placed onto flatsupport membranes or slips of treated glass. A high density grid cancontain many thousands of discrete dots. Grid assays usually involvehybridization with synthetic oligonucleotides, to look for genescontaining specific sequences, or to determine the degree to which aparticular gene is active. Applications include library screening,sequencing by hybridization, diagnosis by hybridization, and studies ofgene expression. High density grids provide the potential for very highthroughput at low cost, if analyzing the grids can be made simple andreliable. Therefore, considerable commercial attention is directed atcompanies developing technology for creating, detecting, and analyzinghigh density arrays of genomic sequences.

Combinatorial assays. Some assays involve small particles (typicallybeads coated with compounds) which act as the reactive sites. Theremight be many thousands of beads, each coated with a different compound(e.g. molecular variants of an enzyme) from a combinatorial library.These beads are exposed to a substance of interest (e.g. a clonedreceptor) in wells, or in a gel matrix. The beads which interact withthe target substance are identified by fluorescence emission orabsorption in the region around each bead. Beads which interact aresurrounded by faint areas of altered luminance. Very sensitive detectorsare required to identify the subtle alterations in luminance around thebeads that interact with the target.

Electrophoretic separations. A solubilized sample is applied to amatrix, and an electrical potential is applied across the matrix.Because proteins or nucleic acids with different amino acid ornucleotide sequences each have a characteristic electrostatic charge andmolecular size, components within the sample are separated bydifferences in the movement velocities with which they respond to thepotential.

The separated components are visualized using isotopic, fluorescent, orluminescent labels. In many cases (e.g. chemiluminescence), theluminance from the specimen is very dim.

Assays which occur within a regularly spaced array of active sites(wells, dot blots within a grid) can be referred to as fixed formatassays. Assays which involve specimens that are irregularly distributedwithin a gel or blot matrix can be termed free format assays.

Fixed format assays are usually performed without imaging. In contrast,free format assays require the use of image analysis systems which candetect and quantify reactions at any position within an image.

Instruments designed for fixed format assays generally lack imagingcapabilities, and have not been applied to free formats. Similarly, veryfew imaging instruments designed for free formats have been applied towells, and other fixed format targets.

Nonimaging Counting Systems

Nonimaging counting systems (liquid scintillation counters,luminometers, fluorescence polarization instruments, etc.) areessentially light meters. They use photomultipliers (PMTs) or lightsensing diodes to detect alterations in the transmission or emission oflight within wells. Like a light meter, these systems integrate thelight output from each well into a single data point. They provide noinformation about spatial variations within the well, nor do they allowfor variation in the packing density or positioning of active sites.

Each PMT reads one well at a time, and only a limited number of PMTs canbe built into a counting system (12 is the maximum in existing countingsystems). Though the limited number of PMTs means that a only few wellsare read at a time, an array of wells can be analyzed by moving the PMTdetector assembly many times.

The major advantages of nonimaging counting systems are that they are a“push-button” technology (easy to use), and that the technology ismature. Therefore, many such instruments are commercially available, andtheir performance is well-characterized.

The major disadvantages of counting systems are:

a. Limited flexibility- few instruments can cope with 384 wells, andhigher density arrays of fluorescent or luminescent specimens are out ofthe question.

b. Fixed format only- designed as well or vial readers, and cannot readspecimens in free format.

c. Slow with dim assays- although scanning a few wells at a time can bevery fast when light is plentiful, dim assays require longer countingtimes at each position within the scan. As there are many positions tobe scanned, this can decrease throughput.

In summary, non-imaging counting systems are inflexible and offerlimited throughput with some specimens.

Scanning Imagers

For flat specimens, an alternative to nonimaging counting is a scanningimager. Scanning imagers, such as the Molecular Dynamics (MD) Storm, MDFluorImager, or Hitachi FMBIO pass a laser or other light beam over thespecimen, to excite fluorescence or reflectance in a point-by-point orline-by-line fashion. Confocal optics can be used to minimize out offocus fluorescence (e.g. the Biomedical Photometrics MACRoscope), at asacrifice in speed and sensitivity. With all of these devices, an imageis constructed over time by accumulating the points or lines in serialfashion.

Scanning imagers are usually applied to gels and blots, where they offerconvenient operation. A specimen is inserted and, with minimal userinteraction (there is no focusing, adjusting of illumination, etc.), thescan proceeds and an image is available. Like the nonimaging countingsystem, the scanning imager is usually a push-button technology. Thisease of use and reasonably good performance has lead to an increasingacceptance of scanning imagers in gel and blot analyses.

Scanning imagers have four major shortcomings:

a. Slow scanning. The beam and detector assembly must be passed over theentire specimen, reading data at each point in the scan. Scanning asmall specimen could easily take 5-10 minutes. A large specimen mighttake ½ scan. This slow scan limits throughput, and complicates thequantification of assays that change during the scan process.

b. Limited number of wavelengths. A limited number of fluorescenceexcitation wavelengths is provided by the optics. Therefore, only alimited number of assay methods can be used.

c. Low sensitivity. Most scanning imagers exhibit lower sensitivity thana state of the art area imager.

d. Not appropriate for luminescence. Scanning imagers require a brightsignal, resulting from the application of a beam of light to thespecimen. Therefore, specimens emitting dim endogenous luminescence(e.g. reactions involving luciferase or luminol) cannot be imaged.

e. Not appropriate for wells. Only flat specimens can be imaged. Alimited number of confocal instruments can perform optical sectioningand then reconstruct the sections into a focused thick image.

Area Imaging

An area imaging system places the entire specimen onto a detector planeat one time. There is no need to move PMTs or to scan a laser, becausethe camera images the entire specimen onto many small detector elements(usually CCDs), in parallel. The parallel acquisition phase is followedby a reading out of the entire image from the detector. Readout is aserial process, but is relatively fast, with rates ranging fromthousands to millions of pixels/second.

Area imaging systems offer some very attractive potential advantages.

a. Because the entire specimen is imaged at once, the detection processcan be very quick.

b. Given an appropriate illumination system, any excitation wavelengthcan be applied.

c. Luminescence reactions (emitting light without incident illumination)can be imaged, including both flash and glow bioluminescence orchemiluminescence.

d. Free or fixed format specimens can be imaged.

Luminescence imaging is more easily implemented, in that illuminationdoes not have to be applied. However, most luminescence reactions arequite dim, and this can make extreme demands upon existing area imagingtechnology. The standard strategy is to use sensitive, cooled scientificgrade CCD cameras for these types of specimens. However, in the absenceof the present invention, integrating cameras will fail to image manyluminescent specimens. Therefore, the present invention can imagespecimens that other systems cannot.

Typical prior art systems apply area imaging to luminescent assays onflat membranes and luminescent assays in wells. Standard camera lensesare always used. The results of well imaging are flawed, in that thereis no correction for parallax error.

There is more extensive prior art regarding use of area imaging influorescence. Fluorescence microscopy (see Brooker et al. U.S. Pat. No.5,332,905) and routine gel/blot imaging are the most commonapplications. Prior art in microscopy-has little relevance, as noprovision is made for imaging large specimen areas.

The existing art relating to macro specimens is dominated by low costcommercial systems for routine gel/blot fluorescence. These systems canimage large, bright areas using standard integrating CCD cameras.However, they have major disadvantages:

a. Limited to the wavelengths emitted by gas discharge lamps. Typicallysome combination of UVA, UVB, UVC, and/or white light lamps is provided.Other wavelengths cannot be obtained.

b. Wavelengths cannot be altered during an assay. If the illuminationmust be changed during the assay (e.g. as for calcium measurement withfura-2), the devices cannot be adapted.

c. Insensitive to small alterations in fluorescence. Transilluminationcomes from directly below the specimen into the detector optics.Therefore, even very good filters fail to remove all of the directillumination, and this creates a high background of nonspecificillumination. Small alterations in fluorescence (typical of many assays)are lost within the nonspecific background.

d. Inefficient cameras and lenses. A very few systems usehigh-performance cameras. Even these few systems use standard CCTV orphotographic lenses, which limit their application to bright specimens.

e. Parallax error precludes accurate well imaging. As fast, telecentriclenses have not been available, these systems exhibit parallax errorwhen imaging wells.

Novel features of the present invention minimize the disadvantages ofknown macro fluorescence systems. These novel features include:

a. Illumination wavelengths may be selected without regard to thepeak(s) of a gas discharge lamp or laser.

b. Using a computer-controlled filter wheel or other device,illumination may be altered during an assay,

c. Small alterations in fluorescence emission can be detected. Becausefluorescence illumination comes via epi-illumination, or from a dorsalor lateral source, direct excitation illumination does not enter theoptics. This renders the nonspecific background as low as possible.

d. Very efficient camera and lens system allow use with dim specimens.

e. Unique telecentric lens is both very fast, and removes parallax errorso well plate assays are accurate.

A primary advantage of the present invention is its fast, telecentriclens, which can image an entire well plate at once, and which canprovide efficient epi-illumination to transparent or opaque specimens.Fiber optic coupling to the specimen can be used instead of lenscoupling. For example, a fiber optic lens has been used with an imageintensified CCD camera run in photon counting mode for analyses of datain fixed or free formats. This approach yields good sensitivity, but hasthe following major disadvantages:

a. Although it is suggested that the system could be used withfluorescent specimens, it would be limited to specimens that aretransilluminated, because there is no place to insert anepi-illumination mechanism. Therefore, the fiber lens system would havedegraded sensitivity, and could not be used with opaque specimens. Manyspecimens are opaque (e.g. many well plates, nylon membranes).

b. Well plates are 8×12 cm. Image forming fiber optics of this size arevery difficult and expensive to construct. Therefore, the specimen wouldhave to be acquired as a number of small images, which would then bereassembled to show the entire specimen.

This multiple acquisition would preclude use of the device with assayswhich change over time.

An area imaging analysis system (LUANA) is disclosed by D. Neri et al.(“Multipurpose High Sensitivity Luminescence Analyzer”, Biotechniques20:708-713, 1996), which uses a cooled CCD, side-mounted fiber opticilluminator, and an excitation filter wheel to achieve some functionssimilar to the present invention (selection of wavelengths, areaimaging). However, LUANA uses a side-mounted fiber optic, which iswidely used in laboratory-built systems, and creates problems that areovercome by the present invention. Specifically, use of a side-mountedfiber optic provides very uneven illumination, particularly when usedwith wells. The epi- and transillumination systems of the presentinvention provide even illumination of both flat specimens and wells.Further, in LUANA, parallax would preclude imaging of assays in wells.

Another system (Fluorescence Imaging Plate Reader-FLIPR of NovelTechInc., Ann Arbor Mich.) uses an area CCD to detect fluorescence within 96well plates. This device is a nonimaging counting system, and uses thearea CCD instead of multiple PMTs. To achieve reasonable sensitivity, itruns in 96 well format and bins all pixels within each well into asingle value. The device is not applicable to luminescence imaging, freeformat imaging, or higher density well formulations and is very costly.

There is extensive prior art in the use of imaging to detect assaysincorporated within microfabricated devices (e.g. “genosensors”). Somegenosensors use scanning imagers, and detect emitted light with ascanning photomultiplier. Others use area CCDs to detect alterations atassay sites fabricated directly onto the CCD, or onto a coverslip thatcan be placed on the CCD. Genosensors have great potential when fixedtargets are defined. For example, a chip is fabricated that looks for aspecific sequence of genomic information, and this chip is used toscreen large numbers of blood samples. While highly efficient for itsdesigned sequence, the chip has to contain a great number of activesites if it is to be useful for screening a variety of sequences.Fabrication of chips with many thousands of sites is costly anddifficult. Therefore, the first generation of genosensors will beapplied to screening for very specific sequences of nucleotides.

The inflexibility of the microfabricated device contrasts with thepresent invention, which does not require microfabrication of the assaysubstrate. Instead, the present invention permits assays to be conductedin wells, membranes, silicalized slides, or other environments. Almostany reaction may be quantified. Thus, the present invention could beused as an alternative technology to microfabrication. Because thepresent invention is flexible, and allows almost any chemistry to beassayed, it can be used for all phases of assay development. Theseinclude prototyping, and mass screening. The invention thereforeprovides an alternative to microfabrication, when microfabrication isnot feasible or cost-effective.

Each of the prior art references discussed above treats some aspect ofimaging assays. However, the prior art does not address all of the majorproblems in imaging large specimens at low light levels. The majorproblems in low light, macro imaging are:

a. very high detector sensitivity required;

b. flexible, monochromatic illumination of large areas is required;

c. parallax error must be avoided; and

d. more reliable procedures are needed to find and quantify targets.

Broadly, it is an object of the present invention to provide an imagingsystem for assays which overcomes the shortcomings of prior art systems.It is specifically intended to provide a complete system for the areaimaging of assays in wells and on membranes. It is specificallycontemplated that the invention provide a complete system for the areaimaging of chemiluminescent, fluorescent, chemifluorescent,bioluminescent, or other nonisotopic hybridization assays, includinghigh density dot blot arrays.

It is another object of the invention to image chemiluminescent,fluorescent, chemifluorescent, bioluminescent, or other nonisotopicassays, including combinatorial assays, in free format.

It is an object of the invention to provide software for digitaldeconvolution of the fluorescence image data. Application of thesoftware decreases flare and out of focus information.

It is also an object of the present invention to provide a method andsystem for imaging assays which are flexible, reliable and efficient inuse, particularly with low level emissions.

The present invention provides synergistic combination of detector,lens, imaging system, and illumination technologies which makes it ableto image the types of specimens previously acquired with nonimagingcounters and scanning imagers. In particular, it can be used with fixedor free formats, and with wells or flat specimens. It is able to detectfluorescence, luminescence, or transmission of light.

The features of the invention include that it detects and quantifieslarge arrays of regularly spaced targets, that it detects and quantifiestargets that are not arranged in regular arrays, and that it performsautomated analyses of any number of regularly spaced specimens, fromsmall numbers of large wells to large numbers of very small wells or dotblots.

It is another feature of the invention to provide an area illuminationsystem that: can deliver homogenous monochromatic excitation to anentire well plate or similarly sized specimen, using standard and lowcost interference filters to select the excitation wavelength; and candeliver varying wavelengths of homogenous monochromatic excitation to anentire well plate or similarly sized specimen, under computer control.

A system embodying the invention provides a lens designed specificallyfor assays in the well plate format. This lens is very efficient attransferring photons from the specimen to the CCD array (is fast),preferably contains an epi-illumination system, and can be used withvery dim specimens. The lens is also telecentric. A telecentric lens hasthe property that it peers directly into all points within a well plate,and does not exhibit the parallax error that is characteristic ofstandard lenses.

A preferred system provides a telecentric and fast lens that generatesan even field of epi-illumination, when required. The lens is equippedwith an internal fiber optic illumination system, that does not requirea dichroic mirror. Preferably, the lens is constructed to accept aninternal interference filter used as a barrier filter. Light rayspassing through the lens are almost parallel when they strike thebarrier filter, so that the filter operates at its specified wavelengthand bandwidth tolerance.

It is a feature of the invention that it provides high light gatheringefficiency, whether used with a fast telecenric lens, or standardphotographic lenses.

A preferred system provides a CCD area array camera that has highquantum efficiency (approximately 80%), and high sensitivity (16 bitprecision), so that most specimens can be detected by integrationwithout intensification. Preferably, the system has an integrating,cooled CCD camera which has coupled thereto an optional imageintensifier. In an embodiment intended for extremely low light levels,incident illumination from the specimen is amplified by the intensifier,and the amplified light is accumulated onto the integrating camera overan integration period. At the end of the integration period, the camerais read out to a dedicated controller or imaging apparatus to reproducethe light image. Multiple exposures may be used to increase the dynamicrange of the camera. A light-tight specimen chamber is provided, towhich all illumination and detection components may be mounted, andwhich contains the specimens.

A system in accordance with the invention may incorporate a translationstage (optional), that may be housed within the light-tight chamber andused to move large specimens (e.g. 22×22 cm membranes) past the opticalsystem. The invention controls the stage motion through software, andthat creates a single composite image from the multiple “tiles” acquiredwith the translation stage.

Preferably, the invention provides software control that corrects theshading, geometric distortion, defocus, and noise errors inherent to thecamera and lens system; and that removes as much nonspecificfluorescence as possible, using multiple images created with differentexcitation filters.

In particular, the invention provides software to deconvolve images froma single focal plane, using optical characteristics previously measuredfrom the lens and detector system. It should be appreciated that datafrom multiple focal planes may also be deconvolved.

While the preferred embodiment of the invention uses a high-precision,cooled CCD camera, if cost is a major factor, the present inventioncould be constructed using lower cost integrating cameras. In this case,shorter integration periods can be achieved, with a reduction in imagequality and ultimate sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

Further objects, features and advantages of the invention will beunderstood more completely from the following detailed description of apresently preferred, but nonetheless illustrative embodiment, withreference being had to the accompanying drawings, in which:

FIG. 1 is a schematic illustration of a system in accordance with afirst preferred embodiment (upright) of the invention;

FIG. 2 is a schematic illustration, in side view, of the fast,telecentric lens;

FIG. 3 is a detailed illustration of the optical and mechanicalcomponents of the lens and the emission filter holder;

FIG. 4 is a schematic diagram illustrating a second embodiment of asystem in accordance with the invention useful for extreme low lightapplications, which has an intensifier mounted between the lens and theCCD camera;

FIG. 5 is a schematic illustration of the intensifier;

FIG. 6 is a schematic illustration of the diffuse illumination plate inside view, showing how discrete fiber bundles from the main bundle aretaken to locations within the rectangular fiber holder;

FIG. 7 is a schematic illustration of the diffuse illumination plate intop view, showing how discrete fiber bundles from the main bundle aretaken to an array of channels within the fiber holder;

FIG. 8 is schematic diagram of the CCD camera;

FIGS. 9A and 9B, collectively referred to below as FIG. 9, represent aflow chart illustrating the method utilized for image acquisition andanalysis in accordance with the present invention; and

FIG. 10 is a flow chart illustrating the method utilized for locatingtargets in the process of FIG. 9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Turning now to the details of the drawings, FIG. 1 is a schematicdiagram illustrating a preferred embodiment of an imaging system 1 inaccordance with the present invention. System 1 broadly comprises anillumination subsystem 10, an imaging subsystem 12 provided in anhousing 14, and a control subsystem 16. The imaging subsystem 12comprises a CCD camera subsystem 18 housed within a camera chamber 20 ofhousing 14 and a lens subassembly 22 extending between camera chamber 20and a specimen chamber 24. In operation, illumination subsystem 10provides the necessary light energy to be applied to the specimen withinchamber 24. Light energy emitted by the specimen is transmitted throughlens subsystem 22 to camera 18, where an image is formed and transmittedto the control subsystem 16 for processing. Control subsystem 16comprises a camera control unit 26, which is a conventional unit matchedto the particular camera 18 and a computer 28 which is programmed tocontrol unit 26 and to receive data from camera 18, in order to achieveunique control and processing in accordance with the present invention.

The light source for the illumination subsystem 10 is preferably an arclamp 30. Light from lamp 30 is conducted via a liquid light guide 32 tothe optical coupler or filter wheel 34. The liquid light guide 32 isadvantageous in that it transmits in the UV range, and in that it actsto diffuse the input illumination more than a fiber optic would do.

The optical coupler 34 contains a conventional filter holder (not shown)for standard, one inch diameter interference filters. In the preferredconfiguration, a computer controlled filter wheel is used instead of theoptical coupler. The filter wheel can contain a number of filters, whichcan be rapidly changed under computer direction.

A fiber optic bundle 36 carries illumination from the optic coupler orfilter wheel 34 to within the light-tight specimen chamber 24. Thebundle 36 passes through a baffle 38, which allows it to move up anddown during focusing of the specimen holder. Alternatively, the fiberoptic bundle 40 from the epi-illumination ring light in lens 22 may beconnected to the optical coupler 34.

Three forms of illumination system are described, each fed by a discretefiber bundle. These are a transilluminating plate (42), a ring lightexternal to the lens (not shown), and a ring light 44 internal to thelens (22) that performs epi-illumination.

The transillumination plate is a rectangular chamber 50 (see FIGS. 6 and7), within which the discrete fibers 52 from bundle 51 are separated androtated by 90 degrees so that they point laterally, towards thespecimen. The fibers 52 are distributed within the chamber in such a waythat they minimize shading within the illumination pattern. To this end,a larger number of fibers lie in the peripherally outward portions ofthe chamber than lie at its center.

The rectangular chamber 50 contains a diffusing screen 54, and a quartzglass diffusing plate 56. These diffusing elements take as their inputthe discrete points of light from the fibers 52, and create a homogenousillumination over the surface of the plate 56. The chamber 50 may alsocontain a dark field stop, to allow light to enter the specimen from theside.

The external ring light consists of a ring of optical fibers alignedwith the axis of the lens, with a hole in the center large enough toencircle the lens 22. The working distance of the ring light is matchedto the focus distance of the lens 22.

The internal ring light 44 consists of a ring of optical fibers, mountedwithin and axially aligned with the body of the telecentric lens 22, andbehind its front lens element. A diffuser, polarizer, or other circularelement may be placed at the front of the fiber ring 44.

The specimen well plate is carried within a holder 58 (FIG. 6) that ismounted to the fiber optic chamber 50. The holder 58 grips the wellplate at its edges. The bottom of the holder 58 is empty, so as not toimpede viewing of the wells. The holder 58 is mounted to a jack, whichmoves it in the vertical dimension. By adjusting the jack 60, the holder58 moves relative to the lens 22 and the specimen is focused.

The lens 22 is a fast, telecentric lens. The lens contains an emissionfilter slot 62, which accepts three inch diameter interference filtersfor fluorescence imaging. It contains an internal fiber optic ring light44, positioned behind the front lens element. The lens 22 is mounted tothe camera chamber by a flange 64 (see FIG. 2) at its middle. The backof the lens projects into the camera chamber 20, providing ready accessto the emission filter slot 62 without disturbing the specimen. Thefront of the lens projects into the specimen chamber 24.

The cooled CCD camera 18 is mounted directly to the lens. Because thecamera has its own chamber 20, there is no need for concern regardinglight leakage around the cooling, power and data cables that exit thechamber to the camera control unit.

All control, imaging, and analysis functions are resident within thecomputer 28.

Illumination Subsystem

The standard technology for monochromatic area illumination is to usegas discharge illuminators (e.g. UV light boxes), which can deliverabout 5000 uW/cm² of surface at the emission peaks (usually mercury).The lamps are coated with a filter that limits emission to a specificpeak. Although fairly bright, gas discharge lamps are limited inwavelength to the peaks emitted by the excited gas within the lamp.

Other than gas discharge lamps, very few descriptions of areaillumination exist. The major problems are selection of wavelength, andthat direct entrance of the illuminating beam into the collection opticsdegrades sensitivity. To avoid this, light can be delivered from above,from the side, or via dark field or refraction into the specimen. All ofthese techniques have severe limitations. Side-mounted fiber opticilluminators are uneven. They are also unsuited to wells or othernon-flat specimens, because light enters the specimen at an angle andfails to penetrate deep targets. Refractive or dark field illuminatorsrequire special optical components at the well plate, and cannot be usedwith opaque specimens.

A more flexible area illumination system would use a broad-bandillumination source, and would allow any wavelength of monochromaticillumination to be selected by precision filters (usually interferencefilters). Filters are preferred, because variable monochromators or lowcost tunable lasers lack sufficient light output when diffused overlarge areas.

Mercury or xenon arc lamps are often selected for filter-basedmonochromatic excitation. The advantage of an arc lamp is that itsoutput can be made into a narrow beam that can be passed through a smalland readily available interference filter, before being spread over theentire surface of the specimen. Either a lens or fiber optic may be usedto transmit the monochromatic light from the filter to the specimen.

The present invention is much more flexible than any previous device. Itapplies diffuse transillumination (through the specimen), dorsalillumination (via ring light or other source), or epi-illumination(through the lens) to the entire surface of the specimen.Epi-illumination is preferred, because it usually results in lowerbackgrounds, broader dynamic range, and more linear fluorescenceresponse under real-world conditions. The ability to deliver large areamonochromatic epi-illumination is one critical factor that sets thepresent invention apart from prior art.

The present invention addresses three main problems in illuminationdelivery.

a. Filter availability-Close-tolerance filters (e.g. a 10 nm bandwidthfilter), which are readily available in small sizes, are not availablefor large areas of illumination. This problem is overcome by use ofstandard interference filters.

b. Illumination delivery-Application of even, monochromatic, andselectable illumination over an 8×12 cm area is a feature of the presentinvention. An optical coupler or computer-controlled filter wheelaccepts standard interference filters, and is used to selectwavelengths. The optical coupler or wheel may be attached to a speciallydesigned fiber optic plate for transillumination, to a fiber optic ringor panel light for dorsal illumination, or to a fiber optic illuminationassembly within the lens, for epi-illumination.

c. Intensity-The excitation illumination is spread over a large area(typically 96 cm²). As intensity decreases with the square of theilluminated area, the resulting excitation intensity is very low indeed.In many cases, emitted fluorescence will not be detected with standard,scientific-grade cooled CCD cameras. The very sensitive detector of thepresent invention is capable of imaging the low levels of fluorescenceemitted from-large specimens. For the most extreme low light conditions,the present invention incorporates an optional light amplificationsystem that may be inserted between the lens and the CCD camera (seebelow).

Lens Subassembly

FIG. 2 shows the general arrangement of illumination and filtercomponents within the telecentric lens 22. The lens has mounted withinit a fiber optic ring light 44, which projects monochromaticillumination through the front lens element onto the specimen (leftwardin FIG. 2). The focus plane of the ring light is at B, while the focusplane of the entire lens is in front of that point, at A. Placing thefocus of the ring light at a point beyond the specimen minimizesspecular reflections from the specimen.

The emission filter slot 62 allows insertion of an interference filterthat removes excitation illumination from the incoming rays, leavingonly the fluorescence emitted by the specimen.

FIG. 3 shows best the optical components of the telecentric, macro lens22. The lens has 39 surfaces, and the following characteristics:

Effective focal length 164.436 mm Numerical aperture .443 Magnification0.25

Note that light rays are almost parallel at the emission filter slot 62.This allows the filter to operate at its specified wavelength andbandwidth.

Although the present invention may be used with any lens, the highestsensitivity is available from its specially designed lens. This lens isfast, telecentric, and incorporates the epi-illumination systemappropriate to large specimen formats.

Epi-illumination is a standard technology in fluorescence microscopy,where small areas are illuminated. The most efficient way to illuminatea small area is to place dichroic beam splitter behind the objective. Adichroic beam splitter or mirror is a partially reflective surface thatreflects one wavelength range, while allowing another wavelength rangeto pass through.

On a microscope, illumination enters the dichroic mirror from the side.The mirror is angled to reflect the excitation light down through theobjective toward the specimen. Fluorescence emitted by the specimen(shifted up in wavelength from excitation) is collected by theobjective, which passes it upwards towards the dichroic mirror. Thedichroic mirror is transparent to the emission wavelength, so that thelight proceeds through the dichroic to the detector plane. A differentdichroic is required for each excitation/emission wavelength.

There are major difficulties in applying the standard form ofdichroic-based epi-illumination system to macro imaging.

a. The dichroic mirror must be at least as large as the objective itmust fill. Camera lenses are much larger than microscope objectives, andwould need correspondingly large dichroic mirrors. Dichroic mirrors thislarge are not readily available.

b. In a fast macro lens, it is critical that the back lens element bemounted as close as possible to the CCD. Any increase in the distancebetween the rearmost lens and the CCD markedly reduces the working fnumber and the light-gathering efficiency. Therefore, there is no roomfor a dichroic to be mounted behind the lens.

c. In a normal epi-illumination system, the dichroic reflects excitationthrough the entire lens. For this reason, transmission of excitationillumination is highly subject to the optical characteristics of theglasses used in the lens. Very costly (and difficult to work) quartzglass optics are required for UV epi-illumination. These UV-transparentoptics can be constructed in the small sizes needed for a microscopeobjective, but would be astronomically expensive in the large sizesdescribed for the present invention.

d. Dichroic beam splitters absorb light. Typically, they are 80-90%efficient.

A unique property of the present invention is that no dichroic isnecessary. The telecentric lens is large, so there is room to install anillumination assembly within its body. The illuminator is mounted sothat it shines directly at the front lens element, from behind. Thisilluminates the specimen, without any need of a reflective dichroicmirror. Any stray excitation illumination that is reflected back throughthe lens is removed by the emission barrier filter, located posterior tothe illumination source.

Further, the lens is designed so that only one of the fifteen internallens components resides in front of the internal illuminator. This hasthe advantage that internal flare and reflections are minimized. Ofequal importance, only the front lens needs to be transparent to UV. Asingle UV-transparent lens is costly, but not prohibitively so.

The front element of the lens is calculated so as to focus theillumination source beyond the plane of the specimen. The defocus of theillumination source at the specimen plane minimizes reflections. As manywell plates are constructed of polished plastic, and tend to generatespecular reflections, this is an important feature.

The lens is highly efficient. The collection F/# of the lens is 4.5.This implies a collection solid angle of 0.03891 sr, and a collectionefficiency of 0.03891/4p=0.3096%. The expected transmission value is0.85-0.90, giving an overall collection efficiency of 0.263-0.279%. Incomparison to an F/1.2 photographic lens, the expected improvement withthe present lens is about 340%.

The present lens is telecentric. A telecentric lens is free of parallaxerror. Images of deep, narrow targets, made with standard lenses,exhibit parallax error. Circular targets at the center of the image areseen as true circles. However, the lens peers into lateral targets at anangle. Therefore, these lateral targets are seen as semilunar shapes. Inmany cases, one cannot see the bottom of a well at all. A telecentriclens collects parallel rays, over the entire area of a well plate. Thus,it does not peer into any wells at an angle and is free of parallaxerror.

A critical advantage of the present lens is that the internal beam iscollimated at a position appropriate to the insertion of a barrierfilter. That is, the lens is calculated so that rays are nearlyparallel, at a point about midway in the lens barrel. The lens acceptsan interference filter at this point. The filter serves to removeexcitation illumination, and other nonspecific light. The collimatedbeam at this point is critical, because interference filters must bemounted orthogonal to the incoming illumination. If the incomingillumination is at an angle, the filter exhibits alterations in thewavelengths that it passes. In the present invention, light rays arealmost parallel when they strike the filter, yielding the best possibleperformance.

The telecentric lens has a fixed field of view (about 14.5 cm diameter,in this case) but, if larger specimens need to be imaged, a motorizedtranslation table may be mounted within the light-tight chamber. Thetranslation table moves the specimen relative to the lens, undercomputer control. After each motion, a single “tile” is acquired. Whenthe entire specimen has been imaged, all the tiles are recomposed (bythe software) into a single large image, retaining telecentricity,freedom from parallax error, and high resolution over its entiresurface.

Extreme Low Light Modification

FIG. 4 shows a modification to system of FIG. 1, addition of an optionalintensifier 70 to provide an alternate system useful for extreme lowlight imaging. In all other respects the system is essentially identicalto that of FIG. 1. The intensifier 70 is mounted between the telecentriclens 22 and the CCD camera 18.

FIG. 5 shows best the intensifier 70 as being of the GEN 3 type, andincluding a photosensitive cathode 72, a microchannel plate (MCP) 74, aphosphor screen 76, and a vacuum sealed body or enclosure 78. The fast,telecentric lens 22 (FIGS. 2,3) is placed in front of this assembly 70.At its output, the lens is focused on an input window of the cathode 72so as to transfer the specimen image thereto. The photosensitive cathode72 is selected to emit electrons in proportion to the intensity of lightfalling upon it. The MCP 74 is positioned within the vacuum sealed body78, between the cathode 72, and the phosphor screen 76 and coupled tothe cathode 72 at each end. The MCP 74 is provided with an array ofsmall diameter MCP channels, each of which is coated with galliumarsenide. The electrons emitted from the cathode 72 are acceleratedalong the MCP channels to the phosphor screen 76. As the electrons fromthe cathode are accelerated along the small diameter channels, theystrike the coated channel walls to produce additional electrons. As themultiplied electrons leave the MCP channels, they strike the phosphorscreen 76 and produce an intensified image of the specimen on an outputwindow. This image is coupled to the CCD 84 element in the camera by alens 80.

It has been found that the use of the Extended Blue GEN 3 imageintensifier is advantageous over other types of intensifiers in that theimage provided on the output screen is sharper, has less shading error,and has less noise than those produced by GEN 1 and GEN 2 intensifiers.It is to be appreciated, however, that as better intensifiertechnologies are developed, they may be incorporated into the presentsystem.

The integrating camera 18 is configured so that the highly amplifiedimage generated on the output window 78 is focused by the intermediatelens 80 onto the CCD element 84. To image low light specimens, the CCDelement 84 of camera 18 integrates for a period. During the integrationperiod, photons from the output window incident to the CCD element 84are stored as negative charges (the signal) in numerous discrete regionsof the CCD element 84. The amount of charge in each discrete region ofthe CCD element 84 is accumulated as follows.

Signal=Incident light×Quantum efficiency×Integration time

The greater the relative intensity of the incident light coming from theintensifier 70, the greater the signal stored in the correspondingregion of the CCD element 84.

For the most extreme low light conditions, as with the scintillationproximity assay, the present invention allows a light amplifier to beinserted between the lens and the CCD camera. In the preferredconfiguration, this light amplifier is an image intensifier.Intensification, as for example, is disclosed in U.S. Pat. No. 5,204,533to Simonet, involves the coupling of an image intensifier to a CCDcamera. The image intensifier typically includes a photocathode, aphosphor screen, and a microchannel plate (MCP) connected between thephotocathode and phosphor screen. Light amplification factors of up toabout 90,000 are possible with this type of device.

With the intensifier inserted into the optical chain, the presentinvention becomes an image intensified CCD (ICCD) camera. In an ICCDcamera, the image is created at three or four planes. At each of theseplanes, there is some loss of quantum efficiency. Therefore, the imageintensifier is operated at high gain to overcome signal losses withinthe optical chain. At very high gain factors, noise and ionic feedbackthrough the MCP become so severe that further improvement of sensitivityis impossible. Even when run at maximum gain, conventional imageintensified CCD cameras are not sensitive enough to image the dimmestspecimens.

Faced with a typical very dim specimen, most ICCD cameras will fail toproduce an image, or will produce a very poor image, in which the targetwill be difficult to discriminate from background, and the true range oftarget intensities will not be rendered. In the worst cases, the targetwill be indiscriminable from background.

Conventional image intensified CCD cameras use an integration periodequal to a single television frame. The short integration period allowsthe intensifier to be used with standard, low-cost video cameras, as forexample, are used in the television industry. In other cases, theintensifier is gated, to use very short integration periods (e.g. 1msec). The use of gating allows the intensifier to be used in a photoncounting mode.

The present invention offers two methods by which intensified light maybe used. The preferred method involves continuous integration of theoutput of the intensifier onto a cooled CCD camera. This method is fastand efficient, but has limited dynamic range. Cooling of theintensifier, or multiple exposures for different times, may be used toimprove the dynamic range. A second method involves looking at shorterperiods of intensifier output, and photon counting. This method is muchslower, but has broad dynamic range. The present invention allows eitherstrategy to be selected, as warranted by the specimen.

Prior art exists for the use of intensified CCD cameras in well plateassay imaging. Martin and Bronstein (1994) and Roda et al. (1996)discuss use of an intensified CCD camera for the imaging ofchemiluminescent specimens. Only bright specimens can be seen. Noprovisions are made for imaging deep wells without parallax error, orfor applying monochromatic excitation to the specimen.

U.S. Pat. No. 4,922,092 (1990) to Rushbrooke et al. discloses the use ofan image intensified CCD camera which is coupled to a special fibreoptic lens. The fibre optic lens consists of bundles which transmitlight between an array of wells and the input of the intensifier. Whilethe invention disclosed by Rushbrooke is free of parallax, and may besuitable for standard 96 or 384 well plates, it would be incapable ofimaging the very high density well arrays addressed by the presentinvention. Further, the invention disclosed by Rushbrooke lacksillumination capabilities. It is also incapable of imaging specimens infree format, because there is space between the input bundles that isnot addressed. By using lens input, as opposed to fiber optics, thepresent invention allows free format imaging.

In sum, the present embodiment of the invention allows the use of anoptional intensifier placed behind the lens, to detect the most extremelow light specimens. When intensified, the device can be run incontinuous integration or photon counting modes.

With the system shown in FIGS. 4 and 5, only the CCD sensor is cooled.This is sufficient for most purposes. It is to be appreciated however,that the intensifier photocathode 72 could also be cooled, therebyimproving the signal to noise ratio of the intensifier. Similarly, theentire photosensitive apparatus (intensifier+CCD) can be cooled.However, cooling the entire photosensitive apparatus has thedisadvantage that the efficiency of the phosphor on the fibre opticoutput window is decreased.

Although a high quality, scientific grade CCD camera can detect about 50photoelectrons incident to the CCD (depending on how we set reliabilityof detection), this is not an accurate indication of performance inimaging luminescent specimens. Real-world performance is complicated bythe emission and collection properties of the entire optical chain, aswell as by the performance of the CCD camera. Therefore, we need to gobeyond the QE of the detector, and examine the transfer efficiency ofthe entire system.

Three factors dominate the transfer efficiency (photoelectronsgenerated/photons emitted) of the detector system. These are the lightcollection efficiency of the lens, the quantum efficiency of the CCDdetector, and the lens transmittance. We can calculate the number ofphotoelectrons generated as follows:

Npe=τ*φ_(detector)*c.e.*N_(photons)

where:

τ is lens transmittance, about 85-90% for our lens

φ is quantum efficiency of the CCD detector, typically about 35-40%, upto 80% in our case, and

c.e. is collection efficiency of lens, less than 0.1% for fastphotographic lenses, about 1.2% in our case.

In a typical scientific grade CCD camera system, using the fastestavailable photographic lens (f1.2), and with a high quality cooleddetector, the CCD will generate 1 photoelectron for about 5,000-10,000photons generated from a point source in the sample.

The lens of the present invention offers a collection efficiency ofabout 0.271%. The efficiency of the CCD detector is about double that ofother CCDs. The result is that the present invention has the theoreticalability to generate one photoelectron for about 500-1000 photonsgenerated from a point source within the sample. This very high transferefficiency allows detection of specimens that cannot be imaged withprior art systems.

In the alternate embodiment of the invention shown in FIGS. 4 and 5, thesystem incorporates an extended blue type of GEN 3 image intensifier.Other types of intensifiers, although less preferred, may also be used.The three major types of intensifier (GEN 1, GEN 2 and GEN 3) differ inthe organization of their components and in the materials of which thecomponents are constructed. In a GEN 1 intensifier, illuminationincident to a photocathode results in emissions at a rate proportionalto the intensity of the incident signal. The electrons emitted from thephotocathode are than accelerated through a high potential electricfield, and focused onto a phosphor screen using electrostatic orproximity focusing. The phosphor screen can be the input window to avideo camera (as in the silicon intensified target camera), or can beviewed directly. GEN 1 intensifiers suffer from bothersome geometricdistortion, and have relatively low quantum efficiency (about 10%).

The GEN 2 intensifiers, like the GEN 3, incorporate a MCP into an imagetube, between the cathode and an anode. The GEN 2 intensifiers aresmaller, lower in noise, and have higher gain than the GEN 1intensifiers. However their quantum efficiency is fairly low (typically<20%), and they tend to suffer from poor contrast transfercharacteristics. In contrast, the GEN 3 intensifier tube has a quantumefficiency of about 30% or higher (needs less gain), and very highintrinsic contrast transfer. With recent versions of the GEN 3, gainlevels are about equal to those of a GEN 2 (ultimate gain levelavailable is about 90,000). Therefore, a GEN 3 intensifier will tend toyield better images than a GEN 2. Where necessary for reasons of cost orspecific design features, other forms of intensifier could be used.Similarly devices with high intrinsic gain (such as electron bombardedback-illuminated CCD sensors) could be used in place of imageintensifiers.

The CCD camera 18 of the present invention could use integration periodslocked to a gated power supply in the image intensifier, with the resultthat the camera could be read out at very short intervals. Using thegating and fast readout feature, and with the intensifier run at highestgain or with a multistage intensifier, the present invention can therebybe operated as a conventional photon counting camera. Thus, the presentsystem can advantageously be used for both direct imaging of faintspecimens, or as a photon counting camera by changing its mode ofoperation from integration to gating.

CCD Camera System

FIG. 8 is a schematic representation of the CCD camera 18. The camera 18includes a CCD element 84 positioned behind a camera aperture. To reducedark noise produced by electrons within the CCD, the CCD element 84 ismounted to a heat sink 88, which in turn is thermally coupled to aPeltier cooling element and liquid circulation system for providingenhanced heat dissipation. The lens is positioned over the aperture tofocus the image on the CCD element 84. The fast, telecentric lens 22(FIGS. 2 and 3) is mounted directly to the camera body by screws, afterremoving the photographic lens mount. Similarly, the image intensifier70 (when present) is mounted directly to the camera body.

Area imaging systems use CCD arrays to form images. Factors whichinfluence the ability of CCD arrays to detect small numbers of incomingphotons include quantum efficiency, readout noise, dark noise, and thesmall size of most imaging arrays (e.g. 2.25 cm²).

Quantum efficiency (QE) describes the ability of the photodetector toconvert incident photons into electron hole pairs in the CCD.Consumer-grade CCDs typically exhibit QE of about 12-15%. Standard,scientific grade cooled CCD cameras exhibit QE of about 40%. A verylimited number of thinned, back-illuminated CCDs can achieve QE of ashigh as 80% at peak detection wavelengths.

Readout noise originates in the output preamplifier of the CCD, whichmeasures the small changes in voltage produced each time the chargecontent of one or more CCD elements is transferred to it. Readout noiseis directly related to the readout rate, and is decreased by use of slowreadout.

Dark noise is produced by thermally generated charges in the CCD. Byincreasing the background level, dark noise decreases dynamic range. Theconstant dark noise level can be subtracted from the image, but darknoise also has a random noise component which cannot be subtracted. Thiscomponent adds to the noise level of the detector. Dark noise isdecreased by cooling the CCD.

The size of the CCD element is related to its ability to storephotoelectrons (known as the well capacity) and, hence, its dynamicrange. The larger each CCD element in the array, the larger the fullwell capacity and dynamic range of that element. A broad dynamic rangeallows the detector to be used for longer exposure times, withoutsaturation, and this enhances the detection of very small signals.Further, the signal to noise performance of larger elements isinherently higher than that of smaller elements. Most area imagingsystems use relatively small CCDs. This results in limited resolutionfor devices in which the discrete CCD elements are large, and limiteddynamic range for devices in which the discrete CCD elements are small.Devices with limited dynamic range cannot achieve 16 bit precision, andmust be used with relatively bright specimens (e.g. fluorescencemicroscopy, UV gels, very bright chemiluminescence).

The present invention incorporates a CCD system which is designed tominimize all of the problems just described. The CCD array is unusuallylarge (6.25 cm²) and efficient (about 80% quantum efficient). The resultis very high detector sensitivity with broad dynamic range (true 16bit). The preferred support electronics include a high-precisiondigitizer, with minimal readout noise. Preferably, the camera is cooledto minimize dark noise.

An electro-mechanical shutter mechanism is additionally provided withinthe camera, for limiting the exposure of the image on the CCD element.Preferably the camera is a thinned, back-illuminated 1024×1024 pixelblack and white camera with asynchronous reset capability, and highquantum efficiency. The camera provides a 16-bit digital signal outputvia digitization circuitry mounted within the camera control unit, andan interface card mounted within the computer. Data from the CCD aredigitized by the camera control unit at the rate of 200,000pixels/second, and transferred directly to the computer memory.

Following the integration period, the CCD camera accepts a trigger pulsefrom the computer to initiate closure of the electromechanical shutter.With the shutter closed, the image is transferred from the CCD to theinternal frame buffer of the computer.

Although this camera could be used without cooling the CCD element,extended periods of integration are achieved by using a CCD camera withan integral cooling element. The effectiveness of integration is limitedby the degree of cooling. With a non-refrigerated liquid cooling device,sensor temperatures of about −50° C. (below ambient) can be achieved. Atthis temperature, dark noise accumulates at a rate of about 7-10electrons/second. This type of cooling has the advantage of low cost andeasy implementation.

It is to be appreciated, however, that longer periods of integration arepossible if refrigerated liquid or cryogenic cooling are employed.

Control Subsystem

The control subsystem 16 comprises, control unit 26 and computer 28.Camera control unit is a computer controllable unit provided by themanufacturer of camera 18 to control the camera. Computer 28 ispreferably a conventional computer running in the Windows® environmentand is programmed to achieve image acquisition and analysis inaccordance with the present invention.

Camera-based imaging systems lack the sort of push-button operation thatis typical of counting or scanning systems. Focusing the camera,adjusting exposure time, and so forth, can all be inconvenient.

In fact, imaging is inherently more complex than counting single targetswithin wells. Nonimaging counting systems have a relatively easy task.They only need to control the scanning process, control internalcalibration, and create a small array of data points representing eachwell. The sequence of steps might be as follows.

a. Calibrate detector against internal standard.

b. Illuminate one well

c. Position a PMT over the illuminated well.

d. Read well.

e. Transfer data to spreadsheet.

f. Illuminate next well and repeat.

An area imaging system has a much more difficult task. Imaging a wellplate might include the following requirements.

a. Provide adequate illumination over the entire plate.

b. Control a high performance camera.

c. Store geometric and density correction factors.

d. Image specimen.

e. Correct geometric and density variation.

f. If necessary, calibrate image to standards within the specimen.

g. Locate each well and quantify intensity.

h. Transfer data to spreadsheet.

These tasks can only be performed if the imaging system is equipped withsoftware that performs functions b-h, above. The present inventionincorporates such software.

In particular, one aspect of the present invention is software whichcorrects for nonspecific background fluorescence by using two images.The first image is made with an excitation filter that excites as littlespecific fluorescence as possible, while exciting nonspecificfluorescence. The second image is made with an excitation filter thatexcites specific fluorescence as much as possible, and as littlenonspecific fluorescence as possible. An optimal specific fluorescenceimage is made by subtracting the nonspecific image from the specificimage.

FIG. 9 is a flow chart illustrating the primary process performed bycomputer 28 in controlling the system 1 and acquiring data therefrom.After initiation of the process, an image of the specimen is acquired atblock 200 using camera 18. Known processes exist for acquiring biasimages of a specimen. Such bias images take into account all significantdistortions and errors introduced by the system itself when an image istaken. Utilizing one of the known methods, a bias image for the specimenis acquired at step 202.

At Step 204, a non-specific image is acquired. This image determines thecontribution of non-specimen components, such as the support substrate,to the image. This step is indicated as optional, since it would only beperformed in the event that the specimen had to be illuminated in orderto acquire the specimen image, in which event some light would also bereflected from non-specimen elements. On the other hand, if the specimenwere the source of the light for the image (as in chemiluminescence),the non-specific image would not be acquired. Similarly, the step atblock 206 is optional, since it involves obtaining a non-specific biasimage.

At block 208, the specimen bias image is removed or subtracted from thespecimen image, and at block 210 the non-specific bias image issubtracted from the non-specific image. This results in two images inwhich bias effects have been compensated. At step 212, the compensatednon-specific image is removed from the compensated specimen image toproduce a working image in which the effects of the specimen areisolated. Those skilled in the art will appreciate that if steps 204 and206 were not performed, steps 210 and 212 would also not be performed.

Following bias removal, various other corrections are provided (e.g. forgeometric warping originating in the lens), using known processes.

At step 214, the operator inputs to the computer the nominal “grid”spacing and “probe template”. The grid spacing is the nominalcenter-to-center spacing of specimen samples on the substrate. The“probe template” is the nominal definition of a single target (e.g. interms of shape and area) corresponding to one dot on a membrane, onewell in a plate, or similar target. Typically the probe template is acircular area, and there is one probe template for each target in thespecimen. A grid is composed of a matrix containing one probe templatefor each of the targets.

Optionally, the operator can also define an array of “anchor points.”The specimen may include an array of thousands of potential samples. Insome instances, a large proportion of these will be populated, and inothers relatively few will. In those instances in which relatively fewsample points are populated, the specimen will include predefined“anchor” points to aid the system locating the probe template positions.In those instances in which a large proportion of the potential samplesites are populated, the samples themselves provide a sufficientpopulation to position the probe templates, and anchor points may beunnecessary.

At block 216, probe templates of the defined size with the defined gridspacing are generated and superimposed over the working specimen image.At this point, the operator can optionally provide a manual adjustmentto the superimposed grid of probe templates, in order to bring them intogeneral alignment with the actual specimens. He could do so, forexample, by utilizing a mouse to shift the entire array then “grabspecific probe templates and center them over the appropriate targets onthe specimen. The operator might, for example, perform a generalalignment by centering the probe templates in the four corners of thegrid over the appropriate targets of the specimen. Although notessential, this manual adjustment will speed and simplify the processingdone by computer 28.

At block 218, a process is performed, described in more detail below, inorder to determine more precise locations for the probe templatesrelative to the actual location of potential targets. At the outset ofthis process, at block 218, a determination is made whether the targetsor anchor points have been adequately identified or defined. If targetshave been well-defined, control is transferred to block 222, where thearray of probe templates is aligned to the defined targets; if not, butanchors have been well-defined, control is transferred to block 220,where the array of probe templates is aligned to the anchors; otherwise,control is transferred to block 224, where the predefined grid spacingand probe template for the array are utilized. It will be appreciatedthat, in some instances, it may be desirable to align the array onanchors and then on targets.

Once the probe templates and targets are aligned, the measurementswithin the individual probe templates are decoded to differentconditions. For example, a probe may be capable of assuming any of nconditions, and the process of block 226 could decode the sample at eachprobe to one of those conditions. The actual process is performed on astatistical basis, and is best understood from a simple example relatingto resolving a binary decision. However, those skilled in the art willappreciate that the process could actually be applied to resolving amultiple condition process. In the simplest case, the binary decision isa “yes” or “no” decision, which could be related to the presence orabsence of a certain condition. In accordance with the process at block226, the actual levels at every probe of the specimen are measured, amean and standard deviation are determined for the set of samples, andthis results in a working statistical distribution. The decoding of a“yes” or “no” could then be done to any level of confidence selected bythe operator. The operator's selection of a level of confidence resultsin the determination of a threshold level (e.g. based upon that levelbeing located a calculated number of standard deviations from the meanon the distribution curve), and any signal above the threshold levelwould be considered a “yes”, while any signal below the threshold levelwould be considered a “no.”

At block 228, a process is performed to generate a report of the arraydata, based upon the process performed at block 226. It is contemplatedthat this may be any form of report writing software which provides theoperator a substantial amount of flexibility in preparing reports of adesired format. Once the reports are generated, the process ends.

Attached as Appendix A is a more detailed discussion of the process ofFIG. 9.

FIG. 10 is a flow chart illustrating the process performed in block 222of FIG. 9.

After initiation of the process, image background and noise areestimated at block 300. At block 302, a determination is made whether agroup alignment of the grid to the array of targets is necessary. Thiscould be done either visually by an operator or by the system. Thepurpose of this test is to determine whether the grid is aligned to thetargets overall. If done by the system, it would be performed by aconventional procedure for testing alignment of two regular patterns ofshapes. If it is determined that adequate alignment of the group exists,control is transferred to block 306.

At block 304, a group alignment is performed. The purpose of thisoperation is to align the probe template grid roughly with therespective targets. The alignment may be done on the basis of the wholegrid or part of the grid selected by the operator. This alignment couldbe done by the process discussed below with respect to block 306 formaximizing ID, except that ID is maximized over the entire grid.

At block 306, a step-wise process is performed within the area of eachindividual probe template to locate that point which yields the maximumintegrated density, ID, within the probe template, given by the formula(1):

ID(x0, y0)=∫_(S(x0, y0)) D(x, y)W(x−x0, y−y0)dxdy  (1)

where:

(x0,y0) is the center point of a probe template;

S(x0,y0) is the probe template area at (x0,y0);

D(x,y) is the density value (e.g. brighteners) at (x,y); and

W(x,y) is a weighting function (e.g. a two-dimensional Gaussian functionwith its maximum value at (0,0)).

This yields an “A location” for each probe template, which is thatlocation that provides the maximum value in formula (1). The probetemplate location prior to block 306 will be referred to as the “Glocation.”

At block 308, a confidence weighting is performed between the A locationand G location, in order to arrive at the final location of the centerof each probe template. The confidence weighting factor for each Alocation is a form of signal-to-noise ratio. That is, the value of ID ateach point is proportional to the ratio between the ID value at thatpoint and the value determined at block 300 for that point. In effectthe weighting factors are utilized to determine the position of theprobe center along a straight line between the A and G locations, withweighting determining how close the point is to the A location.

Although the detailed description describes and illustrates preferredembodiments of the present apparatus, the invention is not so limited.Modifications and variations will now appear to persons skilled in thisart. For a definition of the invention reference may be had to theappended claims.

What is claimed is:
 1. In an area digital imaging system for assays, amethod for extracting targets on a specimen containing an array oftargets that may not be arranged in perfect regularity, comprising thesteps of: defining a matrix of nominal target locations including aprobe template of predefined, two-dimensional size and shape at each ofa plurality of fixed, predefined grid points on the specimen; anddetermining the most probable location of the probe templatecorresponding to a target by: making use of an image of the specimen,deriving a most likely location for a selected target based upon pixelintensity in the image in the vicinity of a nominal target locationcorresponding to the selected target; and using a confidence valueindicative of reliability of the most likely location as a weightingfactor in shifting the location of the probe template from the nominaltarget location towards the most likely location for the selectedtarget.
 2. The method of claim 1, wherein said determining step isperformed iteratively for each target.
 3. The method of claim 1, whereinthe image used in said deriving step is produced by generating a primaryimage of the specimen showing to best advantage the effect of interest,generating a secondary image which shows minimally the effect ofinterest and combining the secondary image with the primary image. 4.The method of claim 1, wherein the specimen is provided with predefinedanchor points, the matrix being initially oriented relative to theactual target locations in the specimen by placing specific probetemplates over one of: the anchor points; and those target points whichare clearly definable.
 5. The method of claim 1 wherein the confidencevalue for a target is determined by the detectability of the target.